This proposal focusses on transcription initiation and elongation by bacterial RNA polymerase (RNAP). Transcription initiation and elongation involve a series of steps: (i) RNAP binds to promoter DNA, yielding an RNAP-promoter closed complex;(ii) RNAP unwinds promoter DNA, yielding an RNAP-promoter open complex;(iii) RNAP synthesizes the first ~8 -15 nucleotides of RNA as an RNAP-promoter initial transcribing complex, using a "scrunching" mechanism, in which RNAP remains stationary on promoter DNA and pulls in adjacent DNA in each nucleotide-addition cycle;and (iv) RNAP breaks its interactions with promoter DNA and synthesizes the remaining nucleotides of RNA as an RNAP-DNA elongation complex, using a "stepping" mechanism, in which RNAP moves forward on DNA in each nucleotide-addition cycle. Each of these steps is a potential target for transcriptional regulators. Understanding transcription initiation, transcription elongation, and transcriptional regulation will require defining the structural transitions in protein and DNA at each step, defining kinetics of structural transitions, and defining mechanisms by which transcriptional regulators affect structural transitions. The proposed work will use ensemble and single-molecule fluorescence resonance energy transfer, single-molecule nanomanipulation, biochemical methods, and genetic methods to address five specific aims: Specific Aim 1 : Determination of the mechanism of scrunching Specific Aim 2: Determination of the role of scrunching Specific Aim 3: Determination of the mechanism of RNAP active-center-cleft loading Specific Aim 4: Detection and analysis of RNAP active-center conformational cycling Specific Aim 5: Determination of the target and mechanism of transcriptional regulators ppGpp and DksA The results will contribute to understanding bacterial transcription and transcriptional regulation, and will contribute to design and synthesis of small-molecule inhibitors of bacterial transcription, for application in antibacterial therapy. Since bacterial RNAP subunits show sequence, structural, and mechanistic similarities to eukaryotic RNAP subunits, the results also will contribute to understanding eukaryotic transcription and transcriptional regulation.

Public Health Relevance

Bacterial RNA polymerase (RNAP) is a molecular machine that carries out reactions essential for bacterial gene expression and bacterial growth. An important class of broad-spectrum antibacterial therapeutic agents functions by inhibiting RNAP. The proposed work will provide information essential for understanding the mechanism of action of RNAP and for rational design of improved and novel broad-spectrum antibacterial agents that function by inhibiting RNAP.